The Essential Role Of eNOS For Hyperoxia-Induced Endothelial Progenitor Cell Mobilization From Bone Marrow Into Circulation

Introduction and Objectives: Hyperoxia stimulates endothelial progenitor cell (EPC) mobilization by a nitric oxide (NO)-mediated mechanism, however, the isoenzyme involved is unknown. Since eNOS is essential for bone marrow release of EPC in response to hindlimb ischemia (tissue hypoxia), we studied the role of eNOS in the non-physiologic NO-mediated release of EPC from bone marrow in response to hyperoxia.
Methods: 4 eNOS knockout (eNOS ^-/-,), 4 C57/B6, and 12 FVB mice had a nafion polymer-coated microelectrode implanted within the femoral marrow cavity to measure continuous real-time changes in bone marrow NO levels (ΔNO, nM) before and during exposure to hyperoxia (hyperbaric oxygen (HBO) approximating FDA-approved clinical protocols). In another 28 mice, circulating EPC (VEGFR2+/CXCR4+) were quantified by flow cytometry. Some mice were pre-treated with the non-specific nitric oxide synthase (NOS) inhibitor, L-NAME.
Results: The rise in bone marrow NO in response to hyperoxia was significantly decreased, but not completely obliterated in eNOS ^-/- mice compared to controls, whereas L-NAME pre-treatment completely inhibited the rise in bone marrow NO (Figure 1A-B). Although substantially increased levels of NO may be induced by hyperoxia in the eNOS^-/- mice (likely by other NOS isoenzymes), EPC are not released into circulation in these mice in response to hyperoxia. Following HBO treatment, circulating EPC increased significantly in control mice but not in eNOS^-/- mice or animals pre-treated with L-NAME (Figure 1C).
Conclusions: eNOS is not the only isoform responsible for marrow NO generation in response to hyperoxia, however, eNOS is the essential isoform for the NO-mediated mobilization of EPCs from marrow into peripheral circulation following exposure to hyperoxia. These findings have important clinical implications for future therapies aimed at improving EPC mobilization.